Intracerebral hemorrhage (ICH), one of the most common types of stroke, affects millions of people each year. During this type of stroke, also called hemorrhagic stroke, blood accumulates in the brain after a blood vessel rupture, causing severe damage.
Microglia, a type of non-neuronal brain cell, and infiltrating macrophages derived from white blood cells known as monocytes, are extremely important for dealing with the consequences of ICH. Both cell types are critical to immune cell defense in the brain. They remove dead cells, debris, and other threats to return the brain to normal function. The critical nature of their role provides methods for analyzing the outcomes and recovery after ICH induced in animal models. Microglia and infiltrating macrophages can be isolated by using approaches such as magnetic-activated cell separation (MACS) coupled with fluorescence-activated cell sorting (FACS). Additionally, microglia can be depleted within the specific brain region by injection of clodronate liposomes.
MACS and FACS are very efficient for isolating microglia and monocyte-derived macrophages from a brain. This approach uses gentle dissociation techniques to separate the cells of interest from myelin protein, red blood cells, and each other. After dissociation, the myelin and red blood cells are removed by using myelin removal beads and centrifugation. The remaining microglia and macrophages are then marked with fluorescent antibodies to be sorted by FACS. Slight pitfalls of MACS and FACS include the pace at which the procedure must be done (within the span of 5-8 hours), and occasionally the number of sorted cells may be small. Additionally, isolation of microglia and macrophages is time dependent as the number of infiltrating macrophages varies at different time points after ICH .
One can deplete microglia by injecting a specific organ or location with liposomes carrying clodronate. In our study, we depleted microglia in the brain’s striatum by injecting it with clodronate liposomes. This compound induces apoptosis (programmed cell death) of microglia by inhibiting its energy production in mitochondria. By eliminating microglia with clodronate liposomes one can study the effects of microglia in brain disease models by determining what occurs in their absence. However, clodronate liposomes modestly affect the function of other cell types within the brain for at least 7 days, altering the microenvironment. Furthermore, selective microglial depletion has been shown to increase the levels of proinflammatory compounds called cytokines, induce activation of brain cells known as astrocytes, and damage blood vessel integrity in the brain .
In summary, MACS combined with FACS and clodronate liposomes are effective methods for isolating or depleting the microglia and monocyte-derived macrophages in brain samples from animal models. These protocols will provide important means by which to study microglia and macrophage function after ICH and other brain diseases.
These findings were described in two 2019 articles, one published in Frontiers in Cellular Neuroscience and the other in Molecular Neurobiology [1, 2]. This work was conducted in Dr. Jian Wang’s lab and was supported by grants from the National Institutes of Health, Johns Hopkins University, and the American Heart Association.
- Li, Q., et al., Expression of Tmem119/Sall1 and Ccr2/CD69 in FACS-Sorted Microglia- and Monocyte/Macrophage-Enriched Cell Populations After Intracerebral Hemorrhage. Frontiers in Cellular Neuroscience, 2019. 12(520). PMID: 30687011 PMCID: PMC 6333739. doi: 10.3389/fncel.2018.00520
- Han, X., et al., Microglial Depletion with Clodronate Liposomes Increases Proinflammatory Cytokine Levels, Induces Astrocyte Activation, and Damages Blood Vessel Integrity. Molecular Neurobiology, 2019. PMID: 30734229. NIHMSID:NIHMS1018578. doi: 10.1007/s12035-019-1502-9